Cancer is the second most common cause of death in the United States, and is estimated to cause 580,350 deaths in 2013 alone. Approximately 90% of these mortalities will result from cancers progressing to the metastatic stage, where cells from the primary tumor begin to enter and travel through the blood stream to form new tumors throughout the body. This process of metastasis is driven by certain proteins called receptors that are present on the surface of cancer cells at high numbers and/or elevated activities relative to normal cells. The abundance of these receptors allows the cancer to rapidly grow, proliferate, and migrate through the body, and thus many have been validated as therapeutic targets for cancer treatment. For example, the notorious urokinase receptor (uPAR) has been a known cancer target for over two decades. uPAR coordinates multiple processes that are essential for metastasis; it is present at elevated levels in virtually all cancers, and high levels are associatd with increased tumor aggressiveness, resistance to chemotherapy, metastasis, and overall low patient survival time. However, despite its importance, no FDA- approved molecules have become available that target this receptor. This is due to the overwhelming complexity of uPAR; it has multiple functional sites that independently coordinate cancer growth and metastasis. The development of a therapeutic that efficiently blocks all functional uPAR sites still represents a critical barrier to progress in the treatment of metastatic cancer. Here, we have outlined a set of objectives aimed to overcome this barrier: Aim 1) use two pre-existing uPAR antagonists to determine the effect of simultaneously targeting the two major functional uPAR sites on cancer progression in vitro. The results will improve scientific knowledge of key uPAR functions that drive cancer growth and metastasis, and will indicate if there is an additive or synergistic effect of targeting multiple functions simultaneously. Aim 2) Improve and chemically link the two antagonists from Aim 1 to generate an antagonist that binds uPAR with ultra-high (picomolar to femtomolar) affinity and blocks all functions that coordinate cancer progression. Libraries of 107 mutants of each antagonist will be generated using error-prone PCR, expressed on the surface of yeast, and screened for binding to uPAR using high throughput (10,000 cells per second) flow cytometry. This powerful technique applies selective pressure to condense millions of years of evolution into just a few months at the lab bench. Subsequently, the two antagonists with the highest affinity will be coupled using a library of 107 different peptide linkers and similarly screened for binding to uPAR. Linked antagonists with the highest affinity will be tested for their ability to inhibit or prevent cancer progression in vitro. Aim 3) The top candidates from Aim 2 wil be tested for treating metastatic cancer in vivo using animal models - a step required by the FDA before human trials can ensue. Our ultimate objective is to continue our collaborations with physician-scientists at Stanford to develop a uPAR-targeted therapy and translate it into the clinic for treatment of metastatic cancer.